Proteinaceous molecules such as enzymes, hormones, storage proteins, binding proteins, transport proteins and signal transduction proteins may be produced and purified using various recombinant DNA techniques. For instance, DNA fragments coding for a selected protein, together with appropriate DNA sequences for a promoter and ribosome binding site are ligated to a plasmid vector. The plasmid is inserted within a host prokaryotic or eukaryotic cell. Transformed host cells are identified, isolated and then cultivated to cause expression of the proteinaceous molecules. One method used to purify hybrid polypeptides is the poly-arginine system in which a hybrid polypeptide is selectively purified on a cation exchange resin. See Sassenfeld, H. M. and Brewer, S. J. BioTechnology, 2:76 (1984); U.S. Pat. No. 4,532,207. Sassenfeld and Brewer reported a carboxy-terminal extension of five arginine residues fused to a target protein. This basic polyarginine extension allowed the purification of the hybrid polypeptide on a SP-Sephadex resin. An analogous protein expression and purification system employs a polyhistidine tract or tag at either the amino- or carboxy-terminus of the hybrid polypeptide. The fusion protein is purified by chromatography on a Ni2+ metal affinity resin. See Porath, J., Protein Expression and Purification, 3:7995 (1992).
Additionally, various affinity purification protocols are currently employed to facilitate the isolation of fusion proteins. Affinity chromatography is based on the capacity of proteins to bind specifically and noncovalently with a ligand. Used alone, it can isolate proteins from very complex mixtures with not only a greater degree of purification than possible by sequential ion-exchange and gel column chromatography, but also without significant loss of activity. Typically, a ligand capable of binding with high specificity to an affinity matrix is chosen as the fusion partner. For example, p-aminophenyl-β-D-thiogalactosidyl-succinyldiaminohexyl-Sephar ose selectively binds to β-galactosidase allowing the purification of β-gal fusion proteins. See Germino et al., Proc. Natl. Acad. Sci. USA 80:6848 (1983). Other expression systems which permit the affinity purification of fusion proteins include fusion proteins made with glutathione-S-transferase, which are selectively recovered on glutathione-agarose. See Smith, D. B. and Johnson, K. S. Gene 67:31 (1988). IgG-Sepharose can be used to affinity purify fusion proteins containing staphylococcal protein A. See Uhlen, M. et al. Gene 23:369 (1983). The maltose-binding protein domain from the malE gene of E. coli has been used as a fusion partner and allows the affinity purification of the fusion protein on amylose resins.
Another method used to detect and isolate proteins is by use of an epitope tag. Epitope tagging utilizes antibodies against guest peptides to study protein localization at the cellular level and subcellular levels. See Kolodziej, P. A. and Young, R. A., Methods Enzymol., 194:508–519 (1991). Using recombinant DNA technology, a sequence of nucleotides encoding the epitope is inserted into the coding region of the cloned gene, and the hybrid gene is introduced into a cell by a method such as transformation. When the hybrid gene is expressed the result is a chimeric protein containing the epitope as a guest peptide. If the epitope is exposed on the surface of the protein, it is available for recognition by the epitope-specific antibody, allowing the investigator to observe the protein within the cell using immunofluorescence or other immunolocalization techniques. Further, fusion proteins labeled with such epitope tags are frequently used for purifying proteins utilizing affinity purification techniques.
Thus, epitope tagging has become a powerful tool for the detection and purification of expressed proteins. See Kolodziej, P. A. and Young, R. A., Methods Enzymol., 194:508–519 (1991). Many types of tags have been used, with c-myc and FLAG® tags being two of the most popular epitopes used. See Evan et al., Mol Cell Biol. 5:3610–3616 (1985). Generally, these epitopes are fused to the amino or carboxy-terminus of the expressed protein making them more accessible to the antibody for detection and less likely to cause severe PATENT structural or functional perturbations.
Fusion proteins having the FLAG® octapeptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 1) at the amino-terminus can be affinity purified on an immuno-affinity resin containing an antibody specific for the octapeptide, See Hopp, T. P., et al. Biotechnology, 6:1204 (1988); Prickett, K. S., et al., BioTechniques, 7:580 (1989); and U.S. Pat. No. 4,851,341. The FLAG® epitope tag has been effectively used to detect and purify protein in mammalian and bacterial systems. The original FLAG® sequence is recognized by two antibodies, M1, M2, and a FLAG® sequence with an initiator methionine attached is recognized by a third antibody, M5. The last five amino acids of the FLAG® sequence is a recognition site for the protease enterokinase, thus, allowing for removal of the FLAG® epitope. The FLAG® epitope has been used in various expression systems for detection and purification of heterologous proteins e.g., in E. coil (Brizzard et al., BioTechniques, 16:730–735 (1994)), Saccharomyces cerevisiae (Lee et al., Nature, 372:739–746 (1994); Prickett et al., BioTechniques, 7:580–589 (1989)), Drosophila (Xu et al., Development, 117:1223–1237 (1993)), Baculovirus (Dent et al., Mol.Cell Biol, 15:4125–4135 (1995); Ritchie et al., Biochem Journal, 338:305–10 (1999)), and mammalian systems (Overholt et al., Clin. Cancer Res., 3:185–191 (1997); Schulte am Esch et al., Biochemistry, 38:2248–2258 (1999)). However, in many mammalian expression systems, protein expression levels are low and effective detection of expressed foreign proteins using established methods can be difficult.
There is therefore a need for an epitope tag and expression system employing such epitope tags which would allow for increased sensitivity and detection of recombinant proteins.